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Competition-Mediated FRET-Switching DNA Tetrahedron Molecular Beacon (CF-DTMB) for Intracellular Molecular Detection Nuli Xie, Jin Huang, Xiaohai Yang, Yanjing Yang, Ke Quan, Min Ou, Hongmei Fang, and Kemin Wang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00593 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Competition-Mediated FRET-Switching DNA Tetrahedron Molecular Beacon (CF-DTMB) for Intracellular Molecular Detection Nuli Xie, Jin Huang,* Xiaohai Yang, Yanjing Yang, Ke Quan, Min Ou, Hongmei Fang and Kemin Wang*

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China.

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Abstract Recently, DNA tetrahedron-based sensors for intracellular detection have attracted more attentions due to many interesting properties, including good structural rigidity, excellent biocompatibility, high resistance to enzymatic degradation, and the ability to enter cells without use of transfection agents. However, the previous designs are still restricted by their lack of accuracy, reliability and generality. Herein, to solve these limitation, we describe self-assembly of the competition-mediated FRET-switching DNA tetrahedron molecular beacon (CF-DTMB), and its applications for intracellular tumor-related mRNA detection. In briefly, the recognition strand is partially complementary to its competitor, which is a hairpin stem-loop structure inserted in one edge of the DNA tetrahedron. In the absence of a target, a long domain of the recognition strand hybridizes with the competitor and makes the hairpin structure open, inducing the two labelled dyes spatially separated (FRET off). However, in the presence of the target, the competitor is substituted for the target to bind with the recognition strand, subsequently leading the formation of a stem-loop structure, which drawing two dyes together (FRET on). The results demonstrate that the current strategy possesses the merits of the previous DNA tetrahedron-based sensors, but also improve the accuracy and reliability. Furthermore, we have also demonstrated our design can become a general strategy for detecting and imaging a variety of other molecules, such as adenosine triphosphate (ATP) in living cells. Therefore, the CF-DTMB can serve as an excellent intracellular molecular detection tool, which is promising to have potential for biological and disease studies. KEYWORDS: DNA nanotechnology, DNA tetrahedron, FRET, molecular beacon, intracellular detection


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The cell, as a complicated microcosmic world, implies variety of significant information with diverse spatiotemporal distribution, including nucleic acids, proteins and small molecules.1,2 Deciphering and analyzing these molecules can provide deep understanding of cellular biological processes, then define human healthy and disease states.3-5 For example, some messenger ribonucleic acids (mRNAs) are regarded as “cancer biomarkers” due to their correlation with disease process and cancer progression.6-8 Therefore, effectively monitoring their abnormal expression has great benefit in early disease diagnosis, and cancer prevention. So far, a lot of methods have been used to detect these valuable biomolecules, including quantitative polymerase chain reaction (qPCR),9,10 western blotting,11-13 enzyme-linked immunosorbent assay (ELISA),14,15 DNA microarray.16,17 However, most of these methods are bulk measurements, which can only provide an average level of cell population and cannot reflect the heterogeneity. And also, they need extract samples from killed cells or tissues, destroying the interdependent symphony of cellular constituents. To solve these issues, amounts of methods for intracellular molecules detection and biological imaging have been developed. Compared with the bulk measurements, intracellular molecules detection can detect target molecules in situ and provide precise insight into cellular environment. But it must confront some challenges.18 Firstly, the probes must be introduced into cells through the plasma membrane, which is quite lipophilic and restricts the transport of various molecules; Secondly, various enzymes in living cells have the ability to degrade the probes, which induce high background, as well as false positive signals; Thirdly, the complicated intracellular fluorescent molecules often interfere the real fluorescent signals from probes, which affects precise detection. In the past three decades, DNA nanotechnology has opened up a new field of bottom-to-up nanofabrication.19-21 Since the pioneered work of Seeman in the early 1980s,22 a series of DNA nanostructures have been constructed with promising functions in DNA machines,23-24 molecular motors,25,26 logic gates,27,28 signal amplifiers,29,30 and network computations.31 The DNA nanostructures have well-defined sizes and shapes, and can be modified with fluorescent dyes for signal output. Importantly, it has been proved that DNA nanostructures with appropriate size are capable of ACS Paragon Plus Environment

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penetrating cells without extra transfection agents.32 So, DNA nanostructures-based sensors have become an ideal choice in the field of intracellular molecular detection and biological imaging. Among various DNA nanostructures, DNA tetrahedron is the simple and attractive pattern. The DNA tetrahedron was firstly assembled by Turberfield.33 It is a kind of 3D nanostructure that exhibits good structural rigidity,

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excellent biocompatibility,35 remarkable cellular penetration and high resistance

to enzymatic degradation when used in cell-related applications,36 and provides spatial orientation of functional ligands on their six edges or four acmes.37 For example, Leong et al. constructed nano-SNEL for mRNA imaging in living cells, through modifying the vertex of DNA tetrahedron with molecular beacon.38 Moreover, Fan et al. assembled a reconfigurable DNA tetrahedron embedded with dynamic sequences responded to different targets.39 It was constructed that a series of DNA-based logic gates functioned well based on conformational change of the DNA tetrahedron. Recently, we reported a DNA tetrahedron-based molecular beacon (DTMB) by integrating molecular beacon into one edge of DNA tetrahedron.40 It retained properties of molecular beacon, and showed effective intracellular mRNA imaging. Despite its good performance, its applications are still hampered by some drawbacks. Firstly, the recognition element is not effectively separated from the signal transduction module, which probably has a significant loss on target binding affinity due to the steric hindrance effect.41 Secondly, this strategy cannot be applied for other molecules if their aptamers cannot bring two termini into close proximity upon binding to target, therefore limiting the versatility.42 Last but not least, as time goes on, the DNA nanostructure probably can be degraded by nuclease, generating a high risk of false positive signal. To solve the aforementioned problems, we have upgraded our previous DTMB design, a competition-mediated FRET-switching DNA tetrahedron molecular beacon (CF-DTMB) for intracellular molecular detection. Our current design involves two important improvements: Firstly, the competition-mediated strand displacement mechanism is employed into our design, which brings at least two obvious advantages: (i) It separate the recognition element from the signal transduction element of DNA tetrahedron. The label-free recognition strand (e.g. aptamer) can decrease great loss in ACS Paragon Plus Environment

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its specificity and affinity; (ii) It expands the versatility for various species of analytes because the recognition element (e.g. aptamer) can freely bind to targets without the requirement of two proximate termini. Secondly, FRET signal transduction mechanism is introduced into the CF-DTMB. The superiority of FRET is that it allows the simultaneous tracking of two emission intensities at different wavelengths in the absence or presence of targets, which can offer some improvements: (i) FRET-based signal output can provide precise detection by effectively eliminating system fluctuation and minimizing the influence of external factors; (ii) The occurrence of FRET only depends on the close proximity of two fluorophores, detaching the risk of false positive signal when the nanostructure is completely destroyed; (iii) The intracellular distribution of probes can be tracked by monitoring donor fluorescence, no matter whether the probes recognize their targets. As a proof of concept, we show the self-assembly and applications of our new probe (CF-DTMB) for intracellular detection and imaging of TK1 mRNA and ATP.

EXPERIMENTAL SECTION Materials. All DNA oligonucleotides were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd (Shanghai, China), and the sequences are shown in Table S1. The adenosine triphosphate (ATP), thymidine 5’-triphosphate(TCP), guanosine 5’-triphosphate (GTP), cytidine 5’-triphosphate (CTP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All aqueous solutions were prepared using ultrapure water (≥18MΩ, Milli-Q water purification system, Millipore). Cell medium RPMI 1640 was obtained from GIBICO (USA). Human hepatocellular carcinoma cell line HepG2, human hepatocellular carcinoma cell line SMMC-7721 and human hepatocyte cell line HL-7702 was obtained from our lab. Instruments. Fluorescent spectra were performed on an F-7000 fluorescence spectrometer (Hitachi, Japan). All cells was incubated by using a Thermo FORMA-3111 CO2 incubator (ThermoFisher, USA). Confocal fluorescence imaging studies and MTT assay were, respectively, ACS Paragon Plus Environment

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performed on an Olympus FV 500 confocal laser scanning microscope (Japan) with an objective lens (100×) and a RT 6000 microplate reader. The Flow Cytometry analysis was gained from Gallios machine (Beckman Coulter, USA). Preparation and Characterization of the CF-DTMB. The CF-DTMB was self-assembled according to a previous protocol.43 For TK1 mRNA detection, five oligonucleotide strands including (1), (2), (3), (4) and (5), were mixed in equimolar in TM buffer (20mM Tris, 50mM MgCl2, and pH 8.0), heated to 95℃ for 5 minutes and then immediately cooled on ice in 1 minutes. For ATP detection, (1), (2), (4), (8) and (9), were used for assembly. For the control CF-DTMB, (1), (2), (4), (8) and (10), was used for assembly. All these sequences can be seen in Table S1. The polyacrylamide gel electrophoresis (PAGE) gel analysis of synthesized DNA tetrahedron and the strand displacement process were implemented by using a 12.5% non-denaturing gel and a 8% non-denaturing gel in 1×TBE buffer (Tris borate-EDTA, pH 8.3) respectively. After running at 200 V for 10 min, the gels were run at a constant voltage of 90 V for 2 h at 4oC. The dynamic light scattering (DLS) characterization of DNA tetrahedron was performed using diluted sample. Fluorescence Analysis. The fluorescence spectrum was determined using F-7000 fluorescence spectrometer. Fluorescence emission intensity of FAM (522 nm) and TAMRA (580 nm) was recorded under an excitation wavelength of 484 nm, and the concentration of CF-DTMB used for the fluorescence calibration curve assays was 50 nM in Tris-Mg buffer (20 mM Tris, 20 mM MgCl2, pH=7.4), then incubated with target of a series of concentrations. The fluorescence signal without target was recorded as background signal. In the selectivity studies, the concentration and other conditions of control groups were brought into correspondence with that of the experiment group. In nuclease stability experiments, the fluorescence intensity of all samples was monitored using time-scan model under the treatment of Exo III (1 U/ml) or DNase I enzyme (0.5 U/ml). All fluorescence experiments were repeated three times.


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Confocal Fluorescence Imaging. All cells were grown in RPMI 1640 medium supplemented with 10% inactivated fetal bovine serum, 100 U/ml 1% penicillin and streptomycin solution Under the condition of 5% humid CO2 at 37°C. For confocal imaging studies, cells were firstly cultured on 35-mm dishes at least 24 h, then the cells were washed by PBS buffer (pH 7.4) three times after incubating with CF-DTMB for 4h. Finally, each dish was examined by confocal laser scanning microscopy (CLSM). The fluorescence images were obtained with FAM in green channel and TAMRA in red channel under 484 nm excitation. Cell Viability Assay. To investigate the cytotoxicity, a standard MTT assay was operated. HepG-2 cells were dispersed with replicate 96-well microplates at a density of 1×106 cells/well. Plates were then maintained at 37°C in 5% CO2 atmosphere for 24 h. Afterwards, the cells were treated with three concentrations of CF-DTMB (0, 50, 100nM) for 6h, 12h, 18h, 24 h, respectively, and 100 µL MTT solutions were then added to each well for 4 h. After removing the remaining MTT solution, 150 µL DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a RT 6000 microplate reader. Flow Cytometry. Samples (5×105 cells/mL) were incubated with the CF-DTMB on plates for 4 hour at 37 ℃. After treatment, cells were detached using Trypsin-EDTA Solution. The solution containing treated cells was centrifuged (2000 rpm, 4 min) and resuspended in PBS for three times. Flow Cytometry was performed using Beckman Coulter Gallios machine by monitoring TAMRA fluorescence. qRT-PCR. Total cellular RNA was extracted from HepG2 cells or L02 cells using Trizol reagent S5 (Sangon Co. Ltd., Shanghai, China) according to the indicated protocol. The cDNA samples were prepared by using the reverse transcription (RT) reaction with AMV First Strand cDNA Synthesis Kit (BBI, Toronto, Canada). qRT-PCR analysis of mRNA was performed with SG Fast qPCR Master Mix (2X) (BBI) on a LightCycler480 Software Setup (Roche). The primers (from 5’ to 3 ’) used in this


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experiment were listed in Table S1. We evaluated all the data with respect to the mRNA expression by normalizing to the expression of GAPDH and using the 2–∆∆Ct method.

RESULTS AND DISCUSSION Working Principle of CF-DTMB for TK1 mRNA detection As shown in Figure 1, the DNA tetrahedron is assembled with four different DNA strands ((1), (2), (3) and (4)) using a simple annealing method, which contains six edges and four faces (Scheme S1 in the Supporting Information). Each strands runs around a triangle to form one of the four faces. Each double-strand edge is composed of two complementary subdomain from every two adjacent strands respectively. On one edge, a hairpin structure is embedded in the subdomain of (3), and fluorescent donor (FAM) and acceptor (TAMRA) are labeled at the two terminus of complementary (4), respectively. Particularly, strand (5), acts as recognition strand, partially hybridizes with the half-stem and loop to makes hairpin structure open, inducing the two labelled dyes spatially separated (FRET off). In this state, only the green (FAM) fluorescence can be observed when shining with the light of 484 nm. However, with the introduction of a target strand (6), the tetrahedron (competitor) is gradually kept away from the recognition strand by competition of target, then guiding the formation of a hairpin structure, which leads two adjacent dyes (FRET on). In this state, the red (TAMRA) fluorescence can be detected with the 484 nm irradiation. Thus, the fluorescence emission ratio of acceptor to donor (A/D) can be used as a signal for quantitation of target sequences.

PAGE and DLS Characterization of the CF-DTMB Successful assembly of the DNA tetrahedron was verified by Polyacrylamide Gel Electrophoresis (PAGE) and Dynamic Light Scattering (DLS). We first investigated the primitive DNA tetrahedron, which was assembled by four strands of various length. As seen in Figure 2(a), the DNA tetrahedron in the lane 8 migrated more slowly due to its huge weight and steric hindrance, compared to single strand ACS Paragon Plus Environment

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and other combinations lacking one or two strands (lane 1-lane 7). The results demonstrated the primitive DNA tetrahedron was successfully assembled. Furthermore, as seen in Figure 2(b), we utilized PAGE analysis to investigate the competition-mediated strand displacement mechanism based on DNA tetrahedron. Com- pared lane 1 with lane 2, there was a new band observed on the top of lane 2 after adding recognition strand (5) into DNA tetrahedron to assemble the CF-DTMB, which showed that recognition strand can hybridized with DNA tetrahedron well. However, after incubating CF-DTMB with DNA target (6), the bands in lane 3 were similar with lane 1 (DNA tetrahedron), but appeared another obvious new band, regarded as the hybridization of recognition strand (5) and DNA target (6). This change could be attributed to the successful reaction of strand displacement. Besides, DLS measurement also revealed the formation of DNA tetrahedron with a mean hydrodynamic diameter of 14.3nm (Figure S1 in the Supporting Information), which was consistent with the theoretical value.

Risk of False Positive Signal (CF-DTMB vs DTMB) The DTMB has remarkable nuclease resistance than general molecular beacon, which has been demonstrated by agarose electrophoresis (Figure S2 in the Supporting Information). It can be attributed to protection of the DNA tetrahedron nanostructures from nuclease attack. Although DTMB could definitely enhance nuclease resistance in the presence of enzymes, it was probably degraded as time went on, then produced false positive signal. In this work, we further want to prove that our upgraded CF-DTMB have better capability of avoiding false positive signal by using both deoxyribonuclese I (DNase I) and exonuclease III (Exo III). DNase I is a potent endonuclease capable of degrading both ssDNA and dsDNA completely to yield 5’-phosphate-terminated polynucleotides,44 whereas Exo III facilitates progressive removal of nucleotides from the 3’-hydroxyl-terminated ends of dsDNA.45 Approximately 3-fold fluorescence signal enhancement in DTMB assay (Figure 3(b)) while treated with 1U/mL DNase I, but almost no change of the A/D ratio in CF-DTMB was observed (Figure 3(a)). Similar results appeared while treated with 1U/mL Exo III. No false positive was observed in


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CF-DTMB incubated with Exo III (Figure 3(c)). In contrast, a gradually increasing false positive signal was detected in DTMB solution (Figure 3(d)), which would be undistinguishable from a true target binding response. It demonstrated that the CF-DTMB, rather than DTMB, possessed the ability to reduce the risk of false positive signal in spite of destroyed DNA tetrahedron. Although CF-DTMB has the same nuclease resistance performance with DTMB (Figure S2 in the Supporting Information), The differences between DTMB and CF-DTMB were that the fluorophore-quencher model gave a signal change as the DNA structure destroyed by enzyme, but the fluorophore donor-acceptor model did not induce the signal change, because the FRET pairs were not brought together by enzymatic attack. This is critically important when the probes would be used in an intracellular environment. In Vitro Performance of the CF-DTMB To evaluate the in vitro performance of the CF-DTMB, we measured its response behavior to different concentrations of synthetic target DNA, instead of mRNA, at 37oC. As can be seen in Figure 4(a), the fluorescence spectrum variation of CF-DTMB was dependent upon adding various concentrations of target DNA. A gradual decrease in FAM fluorescence intensity and a slow increase in TAMRA fluorescence intensity were emerged alone with increasing concentrations ranging from 0-200 nM. It indicated that the CF-DTMB showed excellent FRET signal switching responded to synthetic target DNA. Moreover, the inset plot showed that the relative variation of A/D ratio was more than 4.5 times, and the limitation of detection (LOD) was calculated as 7.6 nM. The LOD of CF-DTMB is slightly higher than that of DTMB in previous work40, which may be attributed to higher fluorescence background and relatively low FRET efficiency. We also investigated selectivity of our probe by analyzing FRET signal responded to one-base mismatched target DNA (7). The results demonstrated the good selectivity performance of CF-DTMB to distinguish specific target, with approximately 2.5-fold change between completely complementary target and one-base mismatched target (Figure 4(b)). Application of Fluorescence Imaging TK1 mRNA in Living Cells


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To be an ideal intracellular probe, non-cytotoxicity is another essential property. We then used HepG2 cells as an example to perform a standard MTT assay. The results showed non-cytotoxicity of the CF-DTMB in living cells (Figure S3 in the Supporting Information), and demonstrated that the CF-DTMB could be applied for intracellular molecular detection. Meanwhile, according to thermodynamic studies (Figure S4 in the Supporting Information), in the absence of DNA target, the CF-DTMB displayed low background until temperature reached to 55oC where the recognition strand was gradually melted from DNA tetrahedron, partially recovering the hairpin structure and resulting in weak FRET. In the presence of DNA target, the ratio of A/D began to drop down when temperature increased from 37oC to 55oC, which can be attributed to melting of the hairpin stem, resulting in the isolation of two fluorophores. Therefore, the CF-DTMB had good signal-to-background ratio to apply in living cells detection at the temperature range (15 oC -37 oC). Next, intracellular detection application of CF-DTMB was further investigated by detecting endogenous TK1 mRNA, which has been proved to be a biomarker related to cell division and tumor proliferation.46,47 HepG2 and HL7702 were used as a group of model cells because previous studies have found that HepG2 cell has extremely high expression level of TK1 mRNA than HL7702 cell. We first optimized the appropriate imaging time of CF-DTMB by investigating different incubation time, it was suggested that 4h was the appropriate imaging time for use in the following studies because the green fluorescence signal reached saturation and red fluorescence began to weaken at 6h and 8h (Figure S5 in the Supporting Information). Then, we utilized CF-DTMB to image TK1 mRNA in HepG2 and HL7702 cells at 4 h. As shown in Figure 5, intense red fluorescence in HepG2 was detected, but no red fluorescence was observed in HL7702, confirming high expression level of TK1 mRNA in HepG2 than HL7702 cells. Furthermore, flow cytometry experiments were performed by monitoring TAMRA fluorescence to validate that HepG2 cell population treated with CF-DTMB indeed displayed higher TK1 mRNA signal than HL7702 cells (Figure S6 in the Supporting Information). Both the confocal imaging and flow cytometry data were in good agreement with the conventional technique qRT-PCR. (Figure S7 in the Supporting Information). Finally, fluorescence co-localization imaging experiments ACS Paragon Plus Environment

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were implemented to observe the intracellular distribution of CF-DTMB (Figure S8 in the Supporting Information). It was indicated that these probes could enter into cytoplasm, where mRNAs existed. Generality of the Strategy Since the recognition element was a label-free oligonucleotides strand and its complementary hairpin sequence can be easily designed, we supposed that our strategy could be as a general strategy for imaging a variety of intracellular molecules. To demonstrate this, we designed another CF-DTMB, for detecting ATP both in tube and in living cells. ATP is a kind of small molecules which is indispensable for cellular function.48 As shown in Figure 6, The CF-DTMB is assembled with five DNA strands ((1), (2), (4), (8) and (9)) using a similar annealing protocol. Particularly, strand (9), a 27nt ATP aptamer49 as recognition strand, partially hybridizes with the half-stem and loop domain to make the hairpin structure of (8) open, inducing the two labelled dyes spatially separated (FRET off). However, with the presence of ATP, the recognition strand binds to ATP and the tetrahedron (competitor) is kept away from the recognition strand by competition of ATP, then guiding the formation of a hairpin structure, which leads two adjacent dyes (FRET on). Thus, the fluorescence emission ratio of acceptor to donor (A/D) can be used as a signal for quantitation of ATP. As increasing the concentration of ATP, the ratio of A/D gradually increased, with a calculated LOD of 10.4 µM (Figure 7(a)). Also, selectivity experiments revealed CF-DTMB had weak response to the analogues of ATP (GTP, CTP, TTP), displaying good specificity (Figure 7(b)). Then, the probes were used for intracellular ATP imaging. As shown in Figure 8, SMMC-7721 cells treated with CF-DTMB showed remarkable intracellular FRET where fluorescence intensity of TAMRA (red) was really conspicuous. But, while substituting ATP aptamer with mismatched complementary strand (10) to assemble the control probe, there was almost no TAMRA fluorescence showed on the red channel, suggesting that it had no response to intracellular ATP. These results demonstrated that our design is suitable for intracellular ATP detection. Overall, the successful detection of nucleic acid (TK1 mRNA) and small molecule (ATP) supported that this


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CF-DTMB design could be used as a versatile strategy to provide a promising mean for intracellular molecules detection. CONCLUSIONS In summary, we designed a CF-DTMB for intracellular molecular detection, including TK1 mRNA and ATP. By introducing the competition-mediated strand displacement mechanism and FRET-switching transduction to the design, this CF-DTMB not only inherits the traditional advantages such as good structural rigidity, excellent biocompatibility, unshackled mobility, high resistance to enzymatic degradation, and the ability to enter cells without use of transfection agents, it owns some improved virtues. (i) The separation between recognition and signal elements can evade any modification of recognition strand and preserve its original binding affinity.42,

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(ii) This independent design of

recognition strand expands the versatility for various species of molecules because it can freely binding to targets without other interference. (iii) FRET can offers precise detection by measuring the fluorescence intensity ratio of acceptor and donor (A/D) and avoiding system fluctuations.50-52 (iv) FRET can eliminate the risk of false positive signal when the nanostructure is probably destroyed.49 (v) The intracellular distribution of probes can be tracked by monitoring donor fluorescence, no matter whether recognized its target. Therefore, the CF-DTMB can serve as an excellent intracellular molecular detection tool, which is promising to have potential for biological and disease studies. ASSOCIATED CONTENT Supporting Information DNA Sequences, assembly mechanism of CF-DTMB, DLS and thermodynamic characterization of CF-DTMB, gel analysis of nuclease resistance, cell viability by MTT assay, confocal imaging by CF-DTMB sensor with time extending, qRT-PCR analysis of Tk1 mRNA and Co-localization analysis of the CF-DTMB (PDF). The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] . * E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21190044, 21205032), the Foundation for Innovative Research Groups of NSFC (21521063), and the Fundamental Research Funds for the Central Universities. REFERENCES (1) Xie, X. S.; Yu, J.; Yang, W. Y. Living Cells as Test Tubes. Science 2006, 312, 228-230. (2) Santangelo, P.; Nitin, N.; Bao, G. Nanostructured Probes for RNA Detection in Living Cells. Ann. Biomed. Eng. 2006, 34, 39-50. (3) Chattopadhyay, P. K.; Gierahn, T. M.; Roederer, M.; Love, J. C. Single-cell technologies for monitoring immune systems. Nat. Immunol. 2014, 15, 128-135. (4) Wills, Q. F.; Livak, K. J.; Tipping, A. J.; Enver, T.; Goldson, A. J.; Sexton, D. W.; Holmes, C. Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments. Nat. Biotechnol. 2013, 31, 748-752. (5) Spiller, D. G.; Wood, C. D.; Rand, D. A.; White, M. R. H. Measurement of single-cell dynamics. Nature 2010, 465, 736-745. (6) Schwarzenbach, H.; Hoon, D. S. B.; Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 2011, 11, 426-437. (7) Sawyers, C. L. The cancer biomarker problem. Nature 2008, 452, 548-552. ACS Paragon Plus Environment

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Figure 1. Working principle of the CF-DTMB for TK1 mRNA detection.


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Figure 2. (a) Validation of DNA tetrahedron by PAGE analysis. Lane 1: (1), Lane 2: (2), Lane 3: (3), Lane 4: (4), Lane 5: (1)+(2), Lane 6: (2)+(3), Lane 7: (1)+(2)+(3), Lane 8: (1)+(2) +(3)+ (4). (b) Validation of the competition-mediated strand displacement based on the CF-DTMB by PAGE analysis, Lane 1: (1)+ (2) +(3)+ (4), Lane 2: (1)+(2) +(3)+ (4) + (5), Lane 3: (1)+(2) +(3)+ (4) + (5)+ (6), Lane 4: (6).


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Figure 3. Comparison between CF-DTMB and DTMB to reduce the risk of false positive signal by DNase I and Exo III. (a) CF-DTMB was treated with 0.5 U/mL DNase I; (b) DTMB was treated with 0.5 U/mL DNase I; (c) CF-DTMB was treated with 1 U/mL Exo III; (d) DTMB was treated with 1 U/mL Exo III.


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Figure 4. (a) In vitro fluorescence profile of CF-DTMB responded to different concentrations of DNA target. Inset plot was calibration curve of A/D ratio as a function of DNA target concentrations. (b) Selectivity studies of CF-DTMB responded between complementary target and one base-mismatched target (100nM each). Inset is a histogram of A/D ratio to different targets.


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Figure 5. (a) Confocal fluorescence images of TK1 mRNA in HepG2 and HL7702 by CF-DTMB. Scale bars are 20 µm.


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Figure 6. Working principle of the CF-DTMB for ATP detection.


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Figure 7. (a) Fluorescence profile of the CF-DTMB responded to different concentrations of ATP. Inset plot was calibration curve of A/D ratio as a function of ATP concentrations. (b) Selectivity studies of CF-DTMB between ATP and other analogues of ATP (GTP, CTP, TTP) (150 µM each). Inset is a histogram of A/D ratio to different targets.


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Figure 8. Confocal fluorescence images of ATP in SMMC-7721 cells by CF-DTMB and control CF-DTMB, respectively. Scale bars are 20µm.


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